1. Field of the Invention
The present invention relates generally to methods of inhibiting angiogenesis in a patient by administering an effective angiogenesis-inhibiting amount of a thrombin inhibitor, and to the treatment of disease states that result from uncontrolled cell proliferation by administering a thrombin inhibitor alone or co-administering a thrombin inhibitor with an anticancer or cytotoxic agent. Specifically, the thrombin inhibitors used in the methods of the present invention are hirudins.
2. Discussion of the Related Art
The circulatory system serves an important role in the transport of nutrients, proteins, hormones, and other vital molecules that are necessary to maintain life. Blood vessels, which form an intricate network of pathways, represent an integral component of the circulatory system. In mammalian species, the internal surface of a blood vessel lumen is comprised of endothelial cells. These endothelial cells impart a smooth and low resistance quality to the lumenal surface. Critical to the free flow and transport of blood and blood constituents, the smooth and nonadhesive internal surface of the blood vessel increases the ease with which fluid flows. Without a smooth internal surface, blood vessels would become obstructed due to the formation of thrombi or other blockages at “sticky” locations on the internal walls. Complete or even partial blood vessel blockage would cause restriction of blood flow, thereby compromising the viability of living tissue served by the vessel. Thus, endothelial cells represent an important structural component of blood vessels and also provide blood vessels with a smooth internal surface.
The formation of blood vessels in vivo takes place in response to stimuli, which are provided in the form of specialized growth factors. These growth factors induce mitosis in cells already present in blood vessels. The new cells may replace nearby damaged cells, or the new cells may arrange themselves such that new blood vessels are formed. The process of growing blood vessels from endothelial cells is termed “angiogenesis,” which results in, among other characteristics, the vascularization of tissue.
Thrombin may play a role in metastasis and angiogenesis of tumors. Generally, for a tumor to grow larger than a few millimeters in diameter, vascular endothelium must proliferate and form vesicle walls to provide circulation and nutrients to the cells inside of the tumor mass. Thrombin likely potentiates this process by virtue of its ability to induce proliferation of endothelial cells. In addition, thrombin has been shown to disrupt the normal intercellular endothelial cell contacts important in preventing cells and plasma factors from escaping or entering the microvasculature. The present hypothesis that thrombin may increase metastasis by disrupting these contacts is supported by studies demonstrating a correlation between decreased levels of anti-thrombin III (which removes thrombin and other proteases from plasma) and increased tumor metastasis.
Cancerous cells derive from a single cell that has mutated in a way that permits it to escape from the biochemical controls that limit the multiplication of normal cells. Once that cell fails to respond normally to growth inhibitors, it starts to proliferate. When the growing tumor reaches a certain diameter, however, simple diffusion in and out of the tumor tissue no longer suffices to supply oxygen and nutrients and remove waste. Further growth depends on angiogenesis (i.e., the formation of new blood vessels from the existing vascular bed), and the small tumor must produce factors that stimulate the growth of blood vessels. Therefore, the inhibition of angiogenesis, in turn leads to the decrease of proliferation of malignant and neoplastic cells.
Carney, et al., Cell, 15:1341 (1978) have postulated that high-affinity cell surface thrombin receptors may be involved in tumor metastasis and angiogenesis. For example, studies have indicated that thrombin receptors can serve as binding sites for tissue plasminogen activator, a molecule secreted from metastatic tumor cells. Moreover, other studies demonstrate the involvement of tissue plasminogen activator in metastasis and angiogenesis. Thus, many of the effects of plasminogen activator may be mediated through its interaction with the cell surface thrombin receptor. Carney, et al., have therefore proposed that stimulation of the thrombin receptor serves to promote tumor metastases, while inhibition of the receptor will decrease metastatic activity.
Angiogenesis has become a central theme in promoting the understanding of how tissue grows. As indicated above, endothelial cell proliferation is not only desirable, but also necessary to carry out a number of physiological processes, for example the in utero formation of tissues and organs. In other contexts, however, angiogenesis may be harmful to the overall health of an organism. For example, angiogenesis makes tumor growth and metastasis possible by vascularizing the tumor, thereby supplying the tumor with blood and nutrients that are necessary to sustain the tumor's growth, as well as providing routes by which tumor cells can migrate to distant parts of the body. (Folkman Cancer Res. 46(2):467-473 (1986). Clearly then, the prevention or reduction of angiogenesis may be a desirable goal in treating some disorders and diseases. Compounds have been tested for their ability to inhibit or reduce angiogenesis. Inhibitors of vascular endothelial growth factor (VEGF), a protein that selectively induces mitosis of vascular endothelial cells, have been investigated. For example, U.S. Pat. No. 6,284,751 to Aiello, et al., describes using inhibitors of the beta isozyme of protein kinase C to counteract the effects of VEGF. Antibiotics such as minocycline have also been reported to inhibit angiogenesis. Some investigators have reported inhibition of tumor growth as well as reduction in the number of metastatic tumors following administration of minocycline in combination with radiation or chemotherapy. See Tamargo, et al. Cancer Res., 51(2):672-675 (1991), and Teicher, Cancer Res., 52(23):6702-6704 (1992). U.S. Pat. No. 5,843,925 to Backer, et al., describes inhibition of angiogenesis upon administration of certain deoxytetracylines. Many of these angiogenesis-inhibiting compounds, however, have only been tested in vitro for their antiproliferative activity.
Thus, there is a need to identify additional compounds as inhibitors of angiogenesis.